Taeko Kakizawa

Switching from the unfolded to the folded state of the helix-loop-helix domain of the Id proteins based on the O-acyl isopeptide method

The inhibitors of DNA binding and cell differentiation Id1–4 are helix‐loop‐helix (HLH) proteins that negatively regulate DNA transcription by forming inactive dimers with ubiquitous and tissue‐specific bHLH proteins, including E47 and MyoD, respectively. Their highly conserved HLH domains are essential for heterodimerization, but can also self‐associate to highly stable, α‐helix‐rich structures at low micromolar peptide concentrations. Here, we show that the introduction of an O‐acyl isodipeptide unit involving the putative N‐cap serine residue of the C‐terminal helix completely abrogates the propensity of the Id HLH analogue for any secondary and tertiary structure, resulting in a random coil, as shown by CD measurements in nonbuffered aqueous solutions. However, the HLH fold reappears as soon as an O → N intramolecular acyl migration, which occurs spontaneously under physiological conditions, restores the native N‐cap serine residue. These results show that changes addressing the N‐terminus of the C‐terminal helix can dramatically influence the HLH structure, and suggest that local interactions at the junction between the loop and the C‐terminal helix might be crucial during the HLH folding process. Furthermore, the present study contributes to the evaluation of the O‐acyl isodipeptide unit as a powerful tool to introduce a conformational switch into peptides. Copyright

Inhibitory effect of a dimerization-arm-mimetic peptide on EGF receptor activation

A cyclic decapeptide was chemically synthesized that mimics the loop structure of a beta-hairpin arm of the EGF receptor, which is highly involved in receptor dimerization upon activation by ligand binding. This peptide was revealed to reduce dimer formation of the receptor in a detergent-solubilized extract of epidermoid carcinoma A431 cells and to inhibit receptor autophosphorylation at less than 10 microM in the intact cells.

Design, synthesis, and evaluation of Trolox-conjugated amyloid-β C-terminal peptides for therapeutic intervention in an in vitro model of Alzheimer's disease.

Two hallmarks of Alzheimers disease (AD) observed in the brains of patients with the disease include oxidative injury and deposition of protein aggregates comprised of amyloid-β (Aβ) variants. To inhibit these toxic processes, we synthesized antioxidant-conjugated peptides comprised of Trolox and various C-terminal motifs of Aβ variants, TxAβx-n (x=34, 36, 38, 40; n=40, 42, 43). Most of these compounds were found to exhibit anti-aggregation activities. Among them, TxAβ36-42 significantly inhibited Aβ1-42 aggregation, showed potent antioxidant activity, and protected SH-SY5Y cells from Aβ1-42-induced cytotoxicity. Thus, this method represents a promising strategy for developing multifunctional AD therapeutic agents.

Synthesis of amyloid β peptide 1–42 (E22Δ) click peptide: pH-triggered in situ production of its native form

Amyloid beta peptide (Abeta) 1-42 is known to be involved in the onset of Alzheimers disease (AD). We developed a click peptide of Abeta1-42 as a useful tool for AD research on the basis of an O-acyl isopeptide method. The click peptide quickly produced intact Abeta1-42 via a pH-dependent O-to-N intramolecular acyl migration (pH-click). Herein, a click peptide (26-O-acyl isoAbeta1-42 (E22Delta)) of a new mutant Abeta1-42 (E22Delta) was synthesized. The mutant click peptide was more water-soluble than Abeta1-42 (E22Delta). Moreover it quantitatively converted to the native peptide under physiological conditions (pH 7.4, 37 degrees C). CD analyses showed a conformational change from a random-coil structure of the click peptide to a beta-sheet structure of the in situ produced Abeta1-42 (E22Delta). This click peptide is a useful precursor of a mutant Abeta1-42 to establish an experiment system for investigating the properties of the mutant.

Evaluation of superior BACE1 cleavage sequences containing unnatural amino acids

A recombinant form of BACE1 (β-site amyloid precursor protein cleaving enzyme-1) corresponding to positions 46-454 of the extracellular domain of the original membrane enzyme was prepared. The recombinant BACE1 (rBACE1) had the kinetic parameters K(m)=5.5μM and k(cat)=1719s(-1). Using several libraries of substrates containing unnatural amino acids, the specificity of rBACE1 was evaluated. LC/MS of digests derived from the libraries clarified that a dodecapeptide containing unnatural amino acids at P(2) to [Formula: see text] was a superior cleavage sequence.

Development of [Ile40]HTLV-I protease inhibition assay using novel fluorogenic and chromogenic substrate

HTLV‐I is a debilitating and/or lethal retrovirus that causes HTLV‐I‐associated myelopathy/tropical spastic paraparesis, adult T‐cell leukemia and several inflammatory diseases. HTLV‐I protease is an aspartic retropepsin involved in HTLV‐I replication and its inhibition could treatHTLV‐I infection. A recombinant L40I mutant HTLV‐I protease was designed and obtained from Escherichia coli, self‐processingand purification by ion‐exchange chromatography. The protease was refolded by a one‐step dialysis and recovered activity. The cleavage efficiency of the [Ile40]HTLV‐I protease was at least 300 times higher for a fluorescent substratethan that of our previously reported recombinant His‐tagged non‐mutated HTLV‐I protease. In addition, we designed and synthesized a substrate containing a highly fluorescent Mca moiety in the fragment before the scissile bond, and a chromogenic p‐nitrophenylalanine moiety after the scissile bond that greatly amplified spectrometry detection and improved the HTLV‐I protease inhibition potency assay. The HTLV‐I protease inhibition assay with the [Ile40]HTLV‐I protease and fluorogenic substrate requires distinctively less protease, substrate, inhibitor and assay time than our previous methods. This means our new assay is more cost‐effective and more time‐efficient while being reproducible and less labor‐intensive. Copyright

Self-assembly pathways of E22Δ-type amyloid β peptide mutants generated from non-aggregative O-acyl isopeptide precursors.

The recently identified E22Δ-type amyloid β peptide (Aβ) mutants are reported to favor oligomerization over fibrillization and to exhibit more-potent synaptotoxicity than does wild-type (WT) Aβ. Aβ(E22Δ) mutants can thus be expected to serve as tools for clarifying the impact of Aβ oligomers in Alzheimers disease (or Alzheimers-type dementia). However, the biochemical and biophysical properties of Aβ(E22Δ) have not been conclusively determined. Here, we evaluated the self-assembly pathways of Aβ(E22Δ) mutants generated from water-soluble, non-aggregative O-acyl isopeptide precursors. Circular dichroism spectroscopy, Western blot analysis, and thioflavin-T fluorescence intensity and cellular toxicity assays suggest that the self-assembly pathways of Aβ(E22Δ) differed from those of Aβ(WT). Aβ1-40(E22Δ) underwent a rapid random coil→β-sheet conformational change in its monomeric or low-molecular-weight oligomeric states, whereas Aβ1-40(WT) self-assembled gradually without losing its propensity to form random coil structures. The Aβ1-42(E22Δ) monomer formed β-sheet-rich oligomers more rapidly than did Aβ1-42(WT). Additionally, the Aβ1-42(E22Δ) oligomers appear to differ from Aβ1-42(WT) oligomers in size, shape, or both. These results should provide new insights into the functions of Aβ(E22Δ) mutants.

Tetrapeptides, as small-sized peptidic inhibitors; Synthesis and their inhibitory activity against BACE1

β‐Site amyloid precursor protein cleaving enzyme 1 (BACE1) is known to be involved in the production of amyloid β‐peptide in Alzheimers disease and is a major target for current drug design. We previously reported substrate‐based peptidomimetics, KMI‐compounds as potent BACE1 inhibitors. In this study, we designed and synthesized tetrapeptides as low molecular‐sized inhibitors. These exhibited high potency against recombinant BACE1, with the highest IC50 value of 34.6 nM from KMI‐927. Copyright